α1-Adrenoceptor subtype specificity of catecholamine induced transactivation of EGF receptor in CHO cells

Nadir Ulu1,2, Robert H. Henning3, Basak Dalkilic1, Marry Duin3, Sahika Guner1,4, Mehmet Ugur5, Teuta Zoto1, Hakan Gurdal1. 1University of Ankara, Department of Medical Pharmacology, 06100, Ankara, Turkey, 2University of Kirikkale, Department of Medical Pharmacology, 71100, Kirikkale, Turkey, 3University of Groningen, University Medical Center Groningen, Department of Clinical Pharmacology, 9713 AV, Groningen, The Netherlands, 4University of Ufuk, Department of Medical Pharmacology, 06520, Ankara, Turkey, 5University of Ankara, Department of Biophysics, 06100, Ankara, Turkey

Transactivation of epidermal growth factor receptor (EGFR) by α1-adrenoceptor (α1-AR) has been implicated as a major pathway involved in catecholamine induced vascular smooth muscle cells (VSMCs) hypertrophy. We recently demonstrated transactivation of EGFR in VSMC to also convey contraction, i.e. immediate effects of α1-AR stimulation. However, the mechanism of EGFR transactivation is not completely elucidated yet. One of the critical questions related with this mechanism is whether all three α1-AR subtypes are able to transactivate EGFR. Therefore, in this study, we aimed to dissect α1-AR subtype specific differences in EGFR transactivation. To this end, Chinese Hamster Ovary (CHO) cells which stably express only single type of α1-AR subtype (i.e. α1A-AR or α1B-AR or α1D-AR) were transiently transfected with EGFR with Lipofectamine 2000 reagent. Two days later, phosphorylation of EGFR (pEGFR) after 10 min stimulation with phenylephrine (PE, 10 μM) or epidermal growth factor (EGF, 1 nM) was investigated by Western Blot. Additionally, PE-mediated intracellular Ca2+ responses (1 nM - 10 μM) were measured and non-cumulative concentration-response curves were obtained in non-transfected and EGFR transfected CHO cells. Intracellular Ca2+ was measured by fura-2 fluorescence at room temperature (21 ± 2°C). CHO cells were incubated at room temperature for 50 min with 1 μM fura-2 AM for loading and then washed with fresh buffer. Fluorescence was recorded using a PTI Ratiomaster microspectrophotometer and FELIX software. pEGFR bands were measured semi-quantitatively, expressed as % of control and compared by Mann-Whitney U test. SPSS 17.0 for Windows software was used for statistical analysis. Differences were considered significant at P<0.05 (two-tailed). In α1A-AR CHO cells transfected with EGFR, both PE and EGF significantly increased the phosphorylation of EGFR (% increase in pEGFR was 160.0±18.1 and 231.3±9.6 for PE and EGF respectively, n = 16-23 independent experiments). Similarly, PE and EGF significantly increased pEGFR bands in α1B-AR (% increase in pEGFR was 138.3±25.0 and 198.4±35.3 for PE and EGF respectively, n = 4-15 independent experiments) and α1D-AR (% increase in pEGFR was 266.5±77.1 and 748.2±178.8 for PE and EGF respectively, n = 4-14 independent experiments) expressing CHO cells. α1A-AR mediated intracellular Ca2+ increase was the highest compared to α1B-AR and α1D-AR. However, EGFR transfection of CHO cells did not alter the intracellular Ca2+ response mediated by all three α1-AR subtypes. In conclusion, our data showed that all three different α1-AR subtypes transactivate EGFR in CHO cells. Transactivation of EGFR by α1-AR seems independent of the intracellular Ca2+ increase. Our data suggest that augmentation of VSMC contraction by catecholamine induced EGFR transactivation is likely mediated through changes in Ca2+-calmoduline interaction or its downstream processes.

This study was supported by a research grant from The Scientific and Technological Research Council of Turkey (SBAG-109S125).